The present application concerns methods of selecting the most appropriate therapy for patients suffering from cancer. The application is particularly concerned with measuring the resistance of cancer cells to anti-cancer agents.
Although radiotherapy and chemotherapy have been responsible for curing many people of cancer in the latter half of this century, there still remain a large number of tumours which either show little response to treatment, or respond initially only to recur later. In particular, women treated for ovarian cancer with platinating agents often show encouraging initial responses to chemotherapy (which often involves the use of cis-diamminedichloroplatinum (CDDP) as the drug of first choice), but by 5 years after diagnosis, ⅔ of them have succumbed to their disease. Similarly lung cancer patients may respond favourably to combination chemotherapy regimens containing CDDP at the outset of treatment but very few experience long term survival. A better understanding of the mechanisms underlying the responsiveness of cancers to CDDP could help predict which patients are most likely to benefit from CDDP or whether alternative cytotoxic agents such as taxol or different therapies such as radiotherapy might be appropriate. Understanding treatment response mechanisms also holds the possibility of selectively modulating these mechanisms to improve the treatment of human cancer using, for example, CDDP.
It has become increasingly apparent that certain oncogenes and tumour suppressor genes may not only be implicated in carcinogenesis, but can also influence the sensitivity of malignant cells to therapeutic agents. Attempts have therefore been made to use these and other genes to try and predict the therapeutic response of human cancer to the presently available treatment modalities such as radiotherapy and/or cytotoxic chemotherapy. Research up to the present time, however, has generally attempted to only examine the expression of single tumour related genes as methods of predicting therapeutic response. Research in the public domain has suggested that mutations in the p53 tumour suppressor gene, which can be found in around 50% of common cancers such as those of the breast, lung and ovary, are associated with resistance to treatment with cytotoxic drugs or radiation. Despite a considerable body of work, however, there are at present no successful clinical tests by which the detection of mutations in the p53 gene alone can be used to predict with an acceptable degree of certainty whether or not a patients cancer is likely to respond to chemotherapy with, for example, platinating agents or the newer cytotoxic agents such as taxanes (e.g. Paclitaxel (TAXOL)).
The effect of the expression of single genes alone on the response of human cancer cell lines to treatment with cytotoxic drugs such as CDDP (cisdiamminedichloroplatinum) has been studied in human in vitro cell lines because these present a model system relevant to the response of human cancer in the clinic. In particular, they exhibit the range of sensitivities to cytotoxic drugs and ionising radiation usually encountered in the clinic. Discoveries in human in vitro cell lines, therefore, have a strong possibility of being able to be translated into clinically useful tests for how well cancers may be expected to respond to treatment.
The progress of cells through the cell cycle is governed by holoenzymes formed by a combination of proteins called cyclins, whose levels fluctuate throughout the cell cycle, and cyclin dependent kinases (CDKs) which become active when they join with cyclins. The cyclin/CDK complexes can be. inhibited by proteins termed cyclin dependent kinase inhibitors (CDKIs) which include the protein p21 WAF1/CIP1 (p21).
The protein products of the cyclin D1 and B1 genes and their respective cyclin-dependent kinase partners CDK4 and CDK1 have been studied. Cyclin D1 and CDK4 control the progress of cells through the cell cycle checkpoint between G1 and S-phase (the phase of DNA synthesis). Cyclin B1 and CDK1 control the cell cycle checkpoint just before mitosis. The expression of cyclin D1 protein in a series of 16 human cancer cell lines has been shown to be related to their intrinsic resistance to the cytotoxic drug CDDP (Warenius et al., 1996). Cyclin D1 protein levels, however, showed no relationship with radiosensitivity, another treatment modality. The relationship between cyclin D1 and CDDP resistance is not, however, strong enough on its own to provide the basis of clinically useful predictive assays.
Paclitaxel, which is a member of the class of anti-cancer drugs known as taxanes, has been shown clinically to be of benefit when added to treatment with platinating agents in the clinical treatment of ovarian cancer. It has been reported that cells can become more sensitive to Paclitaxel when they lose normal p53 function as a result of infection with human papilloma virus constructs or SV40 virus constructs (Wahl et al, Nature Medicine, vol. 2, No. 1, 72-79, 1996). This is thought to result from increasing G2/M arrest and apoptosis. However, it is not the case that all p53 mutant cancer cells are sensitive to Paclitaxel (TAXOL). Accordingly, based on this correlation on its own these studies have not been able to engender a reliable predictive method for determining a likely effective treatment in specific cases.
Thus, there are no indicators that measuring the mutational status or levels of expression of the protein products of single oncogenes, proto-oncogenes or tumour suppressor genes in human cancer cells would be able to provide the basis of a reliable clinical test for whether clinical tumours were likely to respond to treatment with chemotherapeutic agents, including platinating agents and CDDP.
Although radiotherapy has been responsible for curing many people of cancer in the latter half of this century, there still remain a large number of tumours which either show little response to treatment, or respond initially only to recur later. A better understanding of the mechanisms underlying the responsiveness of cancers to radiotherapy could help predict which patients are most likely to benefit from radiotherapy, and also holds the possibility of selectively modulating these mechanisms to improve the treatment of human cancer using radiotherapy.
The molecular basis of intrinsic radiosensitivity has been under investigation for many years. A considerable body of research has focused on the degree of DNA damage and its subsequent repair as reflected in the incidence of double strand breaks (dsbs) in the DNA (Kelland et al, 1988; Schwartz et al, 1991), the residual damage remaining in the DNA after cellular rejoining of dsbs (Nunez et al, 1995; Whitaker et al, 1995), and the fidelity of DNA repair (Powell and McMillan, 1994). In addition to DNA damage, however, it has become increasingly apparent that certain oncogenes and tumour suppressor genes may not only be implicated in carcinogenesis, but can also influence the sensitivity of malignant cells to ionising radiation.
As a result of this growing evidence of the role of oncogenes and tumour suppressor genes in the sensitivity of malignant cells to therapeutic agents, attempts have been made to use these and other genes to try and predict the therapeutic response of human cancer to the presently available treatment modalities such as radiotherapy and/or cytotoxic chemotherapy. Research up to the present time, however, has generally attempted to only examine the expression of single tumour related genes as methods of predicting therapeutic response. When investigating the relationship between expression of a chosen gene and intrinsic radiosensitivity, consideration has not necessarily been given as to whether other candidate genes than the one selected for study might also have an affect on the outcome of experiments.
Research into the role of individual genes has focused on a number of cell cycle genes and signal transduction genes. Transfection of normal cell lines with dominant oncogenes such as myc and ras (McKenna et al, 1991) has resulted in increased radioresistance even in the absence of detectable changes in the rate of dsb induction (Iliakis et al, 1990). Several other dominant oncogenes including c-fms, v-sis, v-erb-B, v-abl, v-src, v-cot (Fitzgerald et al 1990, Suzuki et al, 1992, Shimm et al, 1992) and c-Raf (Kasid et al, 1989, Pirollo et al, 1989) have also been reported to modulate cellular radiosensitivity in mammalian cells. The potential relevance of these findings to clinical radiotherapy has been emphasised by observations that high levels of Raf-1 (the normal protein product of the c-Raf-1 proto-oncogene) are related to intrinsic radiosensitivity in human in vitro cell lines (Warenius et al, 1994). However, these results are not sufficient alone to determine the sensitivity of a tumour to radiotherapy in a clinical assay.
An additional body of evidence indicates a positive relationship between mutation in the p53 tumour suppressor gene and increased cellular radioresistance in both rodent and human tumour cells (Fan et al, 1994, Radford 1994, Zhen et al, 1995, Xia et al, 1995, Lee and Bernstein 1993) and in normal cells transfected with mutant p53 (mp53) genes (Pardo et al, 1994, Bristow et al, 1994, Kawashima et al, 1995). Research in the public domain has suggested that mutations in the p53 tumour suppressor gene, which can be found in around 50% of common cancers such as those of the breast, lung and ovary, are associated with resistance to treatment with cytotoxic drugs or radiation. Despite a considerable body of work, however, there are at present no successful clinical tests by which the detection of mutations in the p53 gene alone can be used to predict with an acceptable degree of certainty whether or not a patient""s cancer is likely to respond to radiotherapy. A wide disparity of results in clinico-pathological studies comparing tumour response and p53 status leads to the conclusion that at the present time p53 mutation or the over-expression of p53 protein are not sufficient alone to predict whether or not a human cancer is likely to respond to radiotherapy.
A number of reports suggest that oncogenes and suppressor genes may modulate intrinsic radiosensitivity by their influence on the progress of irradiated cells through radiation-induced blocks at cell cycle checkpoints. G1/S delay, mediated by p53 following exposure to ionising radiation has been implicated as an important measure of cell cycle perturbation which correlates with relative radiation sensitivity (Kastan et al, 1991, McIlwrath et al, 1994; Siles et al, 1996). Also, the expression of dominant oncogenes such as myc and ras (McKenna et al, 1991) or SV40 (Su and Little, 1993) has been shown to induce both radioresistance and a concomitant increase in post-radiation delay at the G2/M checkpoint. It has also been shown that the protein product of the normal c-Raf-1 proto-oncogene was related to radiosensitivity in 19 human in vitro cell lines (Warenius et al, 1994). Recently, it has further been shown that in 6 of the above 19 cell lines, the previously observed Raf-1/radiosensitivity relationship was very strong and related to how rapidly cells exited from a radiation-induced block at the G2/M cell cycle checkpoint. Those radiosensitive human cancer cells with increased expression of the normal Raf-1 protein exhibit more rapid exit from a G2/M block induced by 2Gy of radiation than radioresistant cells with low expression of Raf-1 (Warenius et al, 1996). High expression of the Raf-1 protein product of the normal c-Raf proto-oncogene is related to radiosensitivity but has no relationship with resistance to CDDP. The relationship between Raf-1 and radiosensitivity is not, however, strong enough on its own to provide the basis of clinically useful predictive assays. The same is true of other attempts to correlate the effects of single genes to the success of therapies.
Unfortunately, little is known about whether, or how, oncogenes and suppressor genes may interact to influence the radiosensitivity phenotype of human cancer cells. However, transfection experiments using cells from other mammals, such as REF (rat embryo fibroblasts), have demonstrated greater increases in radioresistance in cells expressing dominant plus co-operating oncogenes than expressing the single dominant oncogenes alone (McKenna et al, 1990, Su and Little 1992, Pirollo et al, 1993). Similarly, radioresistance induced in REF cells by transfection with multiply integrated mutant p53-pro193 alleles was much greater when the mutant p53 gene was co-transfected with H-ras (Bristow et al, 1994).
It has been shown more recently (Warenius et al, 1994, 1996) that measuring Raf-1 protein in the context of wild-type p53 provides a correlation which could possibly provide the basis of a predictive assay for radiosensitivity. This relationship was demonstrated by measuring Raf-1 protein using quantitative Western blotting. Western blotting is, however, expensive, time consuming and laborious. Furthermore, it requires large numbers of cells. It is thus impractical as a routine clinical test in this particular case. A clinical assay is preferably capable of measuring protein levels in individual cells, rather than in homogenates of a million or more cells as used in Western blotting. It is also important to be able to distinguish Raf protein expression in tumour cells from that in normal cells. This requires the ability to gate out cells on the basis that they are diploid rather than aneuploid in flow cytometry assays, or the ability to measure Raf protein in individual cells that can be observed histologically on tissue sections where morphological criteria enable regions of tumour to be distinguished from connective tissue, blood vessels infiltrating white blood cells, or area of necrosis.
Unfortunately all available antibodies against Raf cross-react with an irrelevant epitope on a 48 kD molecule, when examined on Western blots (see FIG. 3). Raf-1 is a 72-74 kD molecule and can thus be distinguished and separately measured on Western blotting. Cellular assays for Raf-1 such as flow cytometry or immunocytochemistry would rot, however, be able to distinguish the correct 72-74 kD molecule from the irrelevant 48 kD molecule. The 48 kD protein is unlikely to be a fragment of the 72 kD Raf proto-oncogene because the 48 kD protein is much more abundant than the 72 kD protein on Western blotting.
Thus, on the basis of the above state of the art, at the present time there are no indicators that measuring the mutational status or levels of expression of the protein products of oncogenes, proto-oncogenes or tumour suppressor genes in human cancer cells would be able to provide the basis of a reliable clinical test for whether clinical tumours were likely to respond to drug and/or radiation treatment.
An object of the present invention is to solve the above problems. Accordingly, this invention provides a method for measuring the sensitivity of a cancer cell to an anti-cancer agent, which method comprises testing a sample for the mutational status, expression, and/or function of a negative signal transduction factor (NSTF), and testing the sample for the mutational status, expression, and/or function of a positive signal transduction factor (PSTF), wherein when the method comprises measuring the radiosensitivity of wild-type p53 cancer cells by testing a sample comprising wild-type p53 cells or an extract therefrom for the abundance of Raf-1 protein by Western blotting, an antibody specific to Raf-1 protein is employed.
In the context of this invention a factor includes any gene, molecule, component or product, and in particular such factors which are contained in cells. The steps of testing for the NSTF and testing for the PSTF can be carried out in any order.
Also in the context of the present invention, a NSTF is intended to include a factor which inhibits or arrests the cell cycle, causes cells to withdraw from the cell cycle, and/or causes apoptosis or other cell death thereby inhibiting cell division. Thus the NSTF may be a suppressor and/or a PSTF inhibitor. Examples of NSTFs include p53 (in particular its mutational status), p21 (in particular the level of expression of p21 and/or the abundance of p21 protein), Raf-1 inhibitors, cyclin D1 inhibitors and cyclin dependent kinase inhibitors such as CDK1 inhibitors and CDK4 inhibitors.
References to PSTFs in the present invention are intended to include a transcription factor, an oncogene, a proto-oncogene, a gene which inhibits and/or controls cell cycle division, and/or a cell surface receptor. Thus, PSTFs include Raf-1 (in particular the level of expression of Raf-1 and/or the abundance of Raf-1 protein), cyclin D1 (in particular the level of expression of Cyclin D1 and/or the abundance of Cyclin D1 protein) or a cyclin dependent kinase such as CDK1 or CDK4 (in particular the abundance of such cyclin dependent kinases).
This invention also provides a kit for measuring the sensitivity of a cancer cell to an anti-cancer agent, which kit comprises:
(i) a means for testing a sample for the mutational status, expression, and/or function of a negative signal transduction factor (NSTF), and
(ii) a means for testing a sample for the mutational status, expression, and/or function of a positive signal transduction factor (PSTF).